CN109419804A - A kind of plant source preparation of livestock and poultry viral infection resisting - Google Patents
A kind of plant source preparation of livestock and poultry viral infection resisting Download PDFInfo
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Abstract
The present invention relates to a kind of plant source preparation of livestock and poultry viral infection resisting, in particular to application of the HHT as broad-spectrum antiviral preparation.Present invention firstly discovers that homoharringtonine (HHT) has the antiviral activity of broad-spectrum high efficacy, 7 class, 9 kinds of virus (VSV, NDV, PEDV, TGEV, AIV, HSV-1, PRV, PRRSV, FMDV) infection host cells and animal (chicken, chicken embryo, pig etc.) can efficiently be inhibited, substantially reduce internal animal virus amount, and mitigate infection symptoms, safe and reliable novel biological agent is provided for aquaculture.The present invention for exploitation broad-spectrum high efficacy new antiviral drug provide foundation and may, to ensure that farming and animal husbandry sustainable development and human health provide a kind of new safe and reliable technological means.For developing traditional medicine resource, China's traditional Chinese medicine is carried forward with far-reaching realistic meaning.
Description
Technical field
The present invention relates to biomedicine fields, specifically, being related to a kind of plant source preparation of livestock and poultry viral infection resisting.
Background technique
It is directed to virus infection at present, especially when pathogen, which belongs to, to happen suddenly or is unknown, is not similar to antibiotic one largely
Sample wide spectrum, efficient, safe drugs treatment means, viral chemical and biological weapons have become the huge potential threat of the whole mankind in addition,
These facts all emphasize the necessity and urgency of New-type wide-spectrum anti-virus formulation development.Vaccine is that one kind generally acknowledges feasible treatment
Means once made positive contribution for human health, but also faced some thorny problems and shortcoming, such as narrow spectrum --- and vaccine is only
It is effective for the virus of particular serotype；Simultaneously because the variation of unpredictable virus so that only infect onste it
The development of vaccine could be carried out afterwards；In addition, the vaccine development there are also some viral infectious (such as AIDS) still belongs to sky window
Phase.The development of broad-spectrum antiviral preparation can complement one another with the use of vaccine, and in fact broad-spectrum antiviral preparation research has obtained
The progress that must be attracted people's attention, it is believed that also occupy superiority in the confrontation with all kinds of viral infectious for the mankind at last and make
It is difficult to the contribution substituted.
Broad-spectrum antiviral preparation correlative study in recent years obtains incremental advances, breaches the bottle of the single host of anti-virus formulation
The neck original thinking relatively weak with limitation and wide spectrum class agent activity, part achievement have faced application.Anti-virus formulation is ground
Study carefully based on two aspect designs, first is that from virus infection level, second is that from host cell defense level.From host cell defense level
Anti-virus formulation is studied, i.e., is needed for virus replication by change or elimination but grows not important egg to cell itself
White or molecule, to influence the life cycle of virus, to reach antiviral effect.It, can for the anti-virus formulation of host cell
Truly to achieve the purpose that broad-spectrum antiviral, and a possibility that drug resistance generates can be minimized, therefore become virus
The important research direction in field.
The isolated high cepehalotaxus fortunei from cephalotaxus plant Japan caephalotaxus sinensis for the first time of Powerll R.G. group in 1972
Ester alkali (abbreviation HHT, schematic arrangement are shown in Fig. 1), and have found it with significant anti-leukocythemia liveness (the white blood of L-1210
Sick mouse is with respect to life span extension 42%, and P388 mouse is with respect to life span extension 238%), but limited at that time by shortage of resources
Not furtherd investigate, cephalotaxus plant is China's specialty tree species, has 8 kinds in China, and 4 mutation are mainly distributed on south,
In the 1970s, wide grace of Shanghai Pharmaceutical Inst., Chinese Academy of Sciences horse etc. is from cepehalotaxus fortunei (Cephalotaxusfortunei
Hook a variety of alkaloid components, including cephalotaxine, cepehalotaxus fortunei ester etc., (Chen Ruiting, Hua Ze, the petty official such as Chen Ruiting have been separated in)
Pharmacological research [J] Science Bulletin of refined harringtonine and homoharringtonine, 1980,25 (18): 859-861.) to these
Compound has carried out anticancer activity experiment, the experimental results showed that, HHT has stronger anti-leukocythemia liveness, it can make to dye
Matter pyknosis, karyorrhexis inhibit albumen synthesis.Clinic trial confirms it to acute myeloblastic leukemia, monocytic leukemia etc.
There is significant curative effect, nineteen ninety HHT is included into " Chinese Pharmacopoeia ", is increasingly becoming the common drug of clinical treatment leukaemia.Closely
During the last ten years, people are keen to targeted therapy, and researching and developing targeting anti-tumor preparation energetically has been the tendency of the day, and imantinib is one
The anti-leukemia medicine of the money 2001 specific tyrosine kinase of targeting in U.S.'s listing, its listing hinder HHT in the U.S.
Popularization, but later research discovery imantinib generates drug resistance, solution in a short time in the patient of about one third
Its drug resistance problems become extremely urgent, and new clinical research proves HHT to targeted drugs such as imantinib, Dasatinib
The curative effect of drug resistance patient is prominent, and HHT is made to cause external attention again, therefore, by clinical test, FDA approval in 2012
HHT is for treating acute myeloblastic leukemia, monocytic leukemia.
Some protein synthesis inhibitors early have been reported that, as prevented translation initiation factor compound eIF4E-eIF4G from being formed
Mortifier 4E2Rcat；If cycloheximide CHX is by interfering transposition step to hinder translation process；Lactimidomycin(LTM)
Targeting translation extends and albumen is inhibited to synthesize.The extracorporeal antivirus effect effect of these mortifiers is also obvious, such as recent LTM quilt
Proof significantly inhibits the RNA virus such as dengue fever virus, and problem is, 4E2Rcat, CHX affect host cell synthesis system, but
It is that cytotoxicity is very big, and does not carry out in vivo studies verifying.This seminar is screened based on function assessment, finds high cepehalotaxus fortunei ester
Alkali HHT can efficiently and a variety of virus replications of selective depression, and do not influence host cell proteins translation.In those early years once have been reported that table
Bright, HHT can inhibit hepatitis B and mouse hepatitis virus to infect to a certain extent, and the mechanism of action and interior resisting virus effect are still
It is unclear.
The anodyne Indomethacin and antidepressant Prozac of Food and Drug Administration (FDA) approval have efficient
Antiviral effect, it was reported that both drugs can with the synthesis of certain cell protein (enzyme) of targeted inhibition, and the latter simultaneously
It is the target site of virus.These FDA ratify the pharmacology of drug with safety it has proven convenient that developing the time of antiviral drugs accordingly
It will significantly reduce with expending, therefore open up new direction for new drug development.FDA in 2012 ratifies HHT, therefore develops antiviral
Having very promising prospects for new drug and define.
Summary of the invention
The object of the present invention is to provide a kind of plant source preparations of livestock and poultry viral infection resisting.
Application it is a further object of the present invention to provide HHT as livestock and poultry broad-spectrum antiviral preparation.
In order to achieve the object of the present invention, the present invention provides the alkaloid component for being originated from cephalotaxus plant in preparation livestock and poultry use
Application in broad-spectrum antiviral medicament.
The alkaloid component of the present invention includes but is not limited to HHT (homoharringtonine), cephalotaxine, cepehalotaxus fortunei
Ester etc..It is preferred that HHT.
Virus of the present invention includes but is not limited to Paramyxoviridae, orthomyxoviridae family, coronaviridae, rhabdovirus
Section, herpetoviridae, Picornaviridae and Arteriviridae virus.
In a specific embodiment of the invention, the virus includes NDV (newcastle disease virus), VSV (vesicular stomatitis disease
Poison), PEDV (Porcine epidemic diarrhea virus), TGEV (transmissible gastro-enteritis virus), AIV (avian influenza virus), HSV-1 (people
Herpes simplex virus), PRV (porcine pseudorabies virus), PRRSV (porcine reproductive and respiratory syndrome virus), FMDV (hoof-and-mouth disease
Poison) etc..
The present invention also provides a kind of plant source preparation of livestock and poultry viral infection resisting, active constituent is to plant from Cephalotaxus
The alkaloid component separated in object, preferably HHT.
The present invention also provides a kind of livestock and poultry anti-viral pharmaceutical compositions, and it includes the lifes separated from cephalotaxus plant
Alkaloids ingredient (preferably HHT), and at least one pharmaceutical excipient.
Preparation of the present invention or pharmaceutical composition can be intramuscular injection, the subcutaneous or dosage forms such as oral.
When the use concentration of HHT be 20-300nM when, can efficiently inhibit 7 class, 9 kinds of viruses (VSV, NDV, PEDV, TGEV,
AIV, HSV-1, PRV, PRRSV, FMDV) infection host cell (HeLa, Vero, Marc145, PK15 cell etc.), by 0.01-
1mg/kg weight dosage can efficiently inhibit virus infection host animal (chicken, chicken embryo, pig etc.), and significant mitigate even is eliminated infection
Animal symptom.
The invention has the following advantages that
Present invention firstly discovers that homoharringtonine (HHT) has the antiviral activity of broad-spectrum high efficacy, it can efficiently inhibit 7 sections
9 kinds of virus (VSV, NDV, PEDV, TGEV, AIV, HSV-1, PRV, PRRSV, FMDV) infection host cells of class and animal (chicken, chicken
Embryo, pig etc.), significant mitigate even removes virus in infection animal body, and safe and reliable novel biological agent is provided for aquaculture.
The present invention for exploitation broad-spectrum high efficacy new antiviral drug provide according to and may, to ensure that farming and animal husbandry sustainable development and the mankind are strong
Kandy is for a kind of new safe and reliable technological means.For developing traditional medicine resource, carrying forward China's traditional Chinese medicine has
What far-reaching realistic meaning and modern medicine were studied is primarily upon direction.
Detailed description of the invention
Fig. 1 is homoharringtonine (HHT) schematic arrangement.
Fig. 2 is influence of the HHT to VSV virus virulence in the embodiment of the present invention 1.Wherein, A indicates HeLa cell infection VSV,
2%DMEM culture medium is changed after 0.5MOI, 1.5h, at the same be separately added into cell culture fluid 0,0.005,0.01,0.05,0.1,
10,100,1000 μM of HHT and generally acknowledged broad-spectrum antiviral drug-Ribavirin (Ribavirin), continue culture for 24 hours, freeze thawing
Three times, using the result of cytopathy political reform measurement virus virulence；As it can be seen that the effectively antiviral concentration of HHT is few compared to Ribavirin
1000 times.B indicates that a part is to change 2%DMEM culture medium after HeLa cell infection VSV, 1MOI, 1.5h, while to cell culture
The HHT of 10,50,100nM are added in liquid, continues to collect cell after cultivating 36h, carries out immunoblotting assay, another part is
2%DMEM culture medium is changed after HeLa cell infection VSV, 0.5MOI, 1.5h, while adding 50nM HHT into cell culture fluid, point
Not after infection 8h, for 24 hours, 36h collect cell sample, carry out immunoblotting assay, virus protein antibody used be VSV-G, can
See, HHT significantly inhibits G-protein expression and in dose dependent and time dependence.C indicates HeLa cell infection VSV,
0.5MOI, from left to right respectively before virus infection 2h, simultaneously, infection after 2h, 6h, 8h be separately added into 50nM HHT, continue to train
Cell is collected after supporting 48h, Western blot (virus protein antibody used is VSV-G) is carried out or freeze thawing uses three times
Cytopathy political reform measures virus virulence, it is seen then that 2h, which adds HHT not, before virus infection influences viral protein expression and virus titer, sense
2h, 6h, 8h add 50nM HHT to significantly inhibit viral protein expression and virus titer to dye simultaneously and after infection, and infect while adding
HHT inhibits virus titer effect most significant.
Fig. 3 is influence of the HHT to NDV virus virulence in the embodiment of the present invention 2.Wherein, A indicates HeLa cell infection GFP-
After NDV, 0.1MOI, 1.5h, into cell culture fluid plus activity be followed successively by 5 from low to high, 10,25,50,100nM
HHT.After continuing culture for 24 hours, observed in fluorescence microscope Olympus IX73, with the result of CellSens software image；As it can be seen that
HHT significantly inhibits GFP-NDV infection and in dose dependent.B indicates that HeLa cell infection GFP-NDV 0.1MOI, 1.5h are backward
The HHT of 10,100,500nM are added in cell culture fluid, continues to collect cell after cultivating 36h, carries out Western blot, institute
It is NDV-NP with virus protein antibody, it is seen then that the HHT of 100nM can completely inhibit NP protein expression.C indicates that 24 orifice plates are paved with
HeLa cell is observed referring to the way of Fig. 3 A in fluorescence microscope Olympus IX73, the suppression that statistics HHT infects GFP-NDV
Rate processed, the results showed that HHT is 18nM to the half inhibiting rate of GFP-NDV.D indicates to be inoculated with 11 ages in days with the GFP-NDV of 50PFU
SPF chicken embryo, experimental group injection concentration are respectively the HHT of 0.07,0.1,0.2mg/kg, and 36h, 48h harvest chicken embryo, 4 DEG C of placement 6h
Afterwards, allantoic fluid is collected, hemagglutinative titer is measured, counts virus titer, the results show that compared to experimental group, 0.2mg/kg experimental group
NDV hemagglutinative titer is significantly inhibited, E indicates that the NDV of 10000PFU is inoculated with 40 age in days SPF chickens, experimental group intramuscular injection 0.2mg/kg
HHT, continuous injection 3 days takes chicken whole blood, liver, lung for the 7th day after virus inoculation, and RT-PCR method detects virus protein N in whole blood
MRNA level in-site, statistical data, the results show that HHT significantly inhibits NDV in the intracorporal proliferation of chicken.
Fig. 4 is influence of the HHT to PEDV virus virulence in the embodiment of the present invention 3.Wherein, A indicates Vero cell infection
The HHT of 10,100,300,500nM is added after PEDV, 0.1MOI, 1.5h into cell culture fluid, harvests cell after 48h, carries out
Western blot, virus protein antibody used are PEDV-N, the results showed that, the inhibiting rate of 100nM HHT is 80%.B table
Show Vero cell infection PEDV, 0.1MOI, before PEDV infects 2h, simultaneously, 6h after 2h, infection after infection, trained to cell
In nutrient solution plus after the HHT of 50nM, infection 48h, after washing 3 times with PBS, 4% paraformaldehyde fixes 15min, and PBS is washed 2 times；0.1%
Triton100 acts on 15min, and PBS is washed 2 times；37 degree of closing 1h of 5%FBS confining liquid, 37 degree of the monoclonal antibody of virus protein N
It is incubated for 1h, PBST is washed 5 times；Two 37 DEG C of anti-FITC incubation 1h, PBST are washed 5 times, fluorescence microscope Olympus IX73 observation simultaneously
Count the fluorescence signal of indirect immunofluorescence method detection virus protein N, it is seen then that 2h is added HHT and does not significantly inhibit disease before infecting
Poison duplication, and infect simultaneously, infection after 2h and infection after 6h add HHT to significantly inhibit virus replication.C indicates Vero cell infection
PEDV, 0.1MOI, infection while the HHT for adding 100,200,500nM, after infecting 48h, cytopathy political reform measures virus titer, knot
Fruit shows that 500nM HHT completely inhibits virus replication.D indicate 3 age in days Infection in Piglets PEDV, experimental group with each three groups of control group,
Experimental group HHT Drug level is 0.05mg/kg, and continuous injection adopts whole blood after 2 days, 5 days and carries out RT-PCR detection, is measured in serum
The mRNA level in-site of virus protein N P is proliferated in the piglet the results show that HHT significantly inhibits PEDV.
Fig. 5 is influence of the HHT to HSV-1 virus virulence in the embodiment of the present invention 4.
Fig. 6 is influence of the HHT to AIV virus virulence in the embodiment of the present invention 5.
Fig. 7 is influence of the HHT to PRRSV virus virulence in the embodiment of the present invention 6.
Fig. 8 is influence of the HHT to TGEV virus virulence in the embodiment of the present invention 7.
Fig. 9 is influence of the HHT to FMDV virus virulence in the embodiment of the present invention 8.
Figure 10 is influence of the HHT to PRV virus virulence in the embodiment of the present invention 9.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Routine test involved in following embodiment:
1, the preparation of cell
Used when the passage of HeLa, Vero, PK15, Marc145 cell and contains 10% fetal calf serum and 100U blueness strepto-
The DMEM culture solution of element, DMEM culture solution of the maintaining liquid containing 2% fetal calf serum and 100U mycillin.
NDV infects HeLa cell, and VSV, HSV-1, PEDV, PRV infect vero cell, and TGEV, FMDV infect PK15 cell,
PRRSV infection Marc145 cell.
2, viral proliferation
The cytotoxic proliferation of HSV-1, NDV, VSV, PRV: according to the amount virus inoculation stoste of cell bottle culture solution 1/10, to
Cytopathy (such as NDV forms big plasomidum) is viral using multigelation method harvest up to 75% (generally 48 hours), i.e., will be thin
Born of the same parents' bottle be put into -80 DEG C freeze after place room temperature to partial melting, yawing cell bottle makes attached cell take off wall at this time, be placed again into -
80 DEG C freeze, and 3 times repeatedly, release virus from cell, freeze in -80 DEG C.
The proliferation of NDV embryo toxicity: 9-10 days instar chicken embryos are inoculated with NDV virus stock solution used through allantoic cavity, collect urine after chicken embryo death
Cyst fluid measures hemagglutinative titer and TCID50, in liquid nitrogen cryopreservation after packing.
3, the measuring method of virus titer titre
Cytopathy (cytopathic effect, CPE) is cytopathic effect, refers to viruses into tissues culture cell
The cell degeneration generated after infecting can carry out Viral Quantification using such pathological effect.It is common that virus infection forms cytopathy
Plasomidum (maxicell that i.e. multiple cell aggregations are formed together multicore) and two kinds of plaque (cell detachment formation plaque).Lesion is melted
Conjunction rate is the ratio that sick cell accounts for all cells.
Toxicity test: cell toxicant (VSV, PEDV, PRV) doubling dilution is obtained into cell toxicant suspension, with 96 orifice plate cultures
10 hole μ L/ of cell toxicant suspension is added into each hole, 12 dilutions and 8 repetitions are done, in 37 to single layer for HeLa, Vero cell
Cytopathy, or record lesion fusion rate (lesion fusion rate, that is, lesion are observed after placing 48 hours in DEG C carbon dioxide incubator
Cell accounts for the ratio of all cells), it is repeated 3 times.
Plaque forming unit (Plaque-forming unit, PFU) measurement is that a kind of measurement viral infection is more accurate
Method.By the viral suspension of debita spissitudo be inoculated into growth cell monolayer glass dish or flat bottle in, when viruses adsorption in
After on cell, then it is covered with one layer of semisolid nutrient agar rouge layer dissolved on it, after to be solidified, is incubated for culture.When virus is thin
After duplication proliferation intracellular, each infectious viral particle generates the infection cell lesion an of limitation in cell monolayer,
Lesion is gradually expanded, if being coloured with dimethyl diaminophenazine chloride isoreactivity dyestuff, " plaque " of coloring is shown without in red background, clearly
Chu Kejian.Since each plaque is formed by single virus particle replication, so the titre of viral suspension can use every milliliter of plaque
Unit (PFU) is formed to indicate.With 12 orifice plate Cultivation of Vero to single layer, the training of 900ul serum-free is added into each hole
It supports the first hole of base and 100ul cell toxicant suspension is added, 10 dilutions are done, after placing 48 hours in 37 DEG C of carbon dioxide incubators
Cytopathy is observed, every block of plate stays a hole negative control, that is, is added without virus liquid.Containing of mixing is added in 37 DEG C of culture 1.5h
1% hole agarose DMEM 1ml/, 37 DEG C of culture 72h.4-6h is fixed with 4% formalin 250ul in every hole, and agar is thrown away, from
Water rinses (movement is soft, prevents cell detachment) well；0.5% crystal violet ethanol solution 200ul, dyeing is added in every hole
15min, tap water are cleaned.It selects the cell hole for 20-100 plaque occur to be counted, PFU/ml=plaque number × virus is dilute
Release multiple ÷ inoculation volume (ml).
TCID50Calculation method: virus is done 10 times and is serially diluted, it is thin to be laterally inoculated with single layer by 96 orifice plate cell monolayers of preparation
Born of the same parents' plate, every dilution repeat 3 holes, observe cytopathy, viral dilution of the record higher than 50% and lower than 50% lesion hole daily
Degree, calculating ratio is away from acquisition TCID50As a result.Calculation formula are as follows: (the lesion rate -50% higher than 50%)/(it is higher than 50% lesion rate -
Less than 50% lesion rate)=than away from；Than away from being added with the index of dilution of the virus close to 50% lesion rate to get index.
Blood clotting (HA) test: the hemagglutinin of NDV and AIV virus or virus can selectively make certain or certain several dynamic
The phenomenon that red blood cell of object is aggregated, this agglutination red blood cell referred to as hemagglutination is claimed using the test that this characteristic designs
Hemagglutination test (HA).It is carried out on 96 hole micro-reaction plates, each hole adds 25ul physiological saline from left to right, adds in the 1st hole of left side
25ul virus liquid allantoic fluid inhales 25ul to the 2nd hole, successively doubling dilution to the 11st hole, inhales and abandon 25ul after mixing；12nd
Hole is red blood cell control.1% chicken erythrocyte suspension 25ul successively is added to each hole from right-to-left, vibrates on the oscillator, room temperature
10min starts observation as a result, result observation can be carried out by having precipitated to control wells red blood cell after lower standing.Red blood cell is all solidifying
Collection is sunken to bottom hole, and it is in netted for tiling, and as 100% agglutination, inagglutinable red blood cell is sunken to bottom hole in dotted.With 100% agglutination
Viral greatest dilution be the viral hemoagglutination valence, as one agglutination unit.
Influence of 1 HHT of embodiment to VSV virus virulence
Infect HeLa cell with the VSV of 0.5MOI, be added respectively into cell culture fluid after 1.5h various concentration HHT and
Ribavirin (Ribavirin), freeze thawing three times, measures virus virulence after infection for 24 hours, the results showed that, compared to Ribavirin,
The effectively antiviral concentration of HHT is 1000 times (Fig. 2A) few.
HeLa cell is infected with the VSV of 1MOI, the HHT of various concentration, virus sense is added after 1.5h into cell culture fluid
Cell is collected after dye 36h.Using the expression of Western blot detection virus protein G-protein, the results showed that, HHT significantly inhibits G egg
It is white to express and be in dose dependent.HeLa cell is infected with the VSV of 0.5MOI, adds concentration to be 50nM HHT after 1.5h, exists respectively
8h, for 24 hours, 36h collect cell sample carry out immune-blotting method virus protein G-protein expression, the results showed that, HHT significantly presses down
The expression (Fig. 2 B) of viral G protein processed.
HeLa cell is infected with the VSV of 0.5MOI, respectively the 2h before virus infection, infected simultaneously, 2h, 6h, 8h after infection
50nM HHT is added into cell culture fluid respectively, 48h collects cell after infection, detects virus protein G using Western blot
The expression or freeze thawing of albumen measure virus virulence three times, the results showed that, 2h adds HHT not influence virus protein before virus infection
Expression and virus titer, 2h, 6h, 8h add 50nM HHT to significantly inhibit viral protein expression and virus drop simultaneously and after infection for infection
Degree infects while HHT being added to inhibit virus titer effect most significant (Fig. 2 C).
Influence of 2 HHT of embodiment to NDV virus virulence
HeLa cell is infected with the GFP-NDV (NDV of Green Fluorescent Protein) of 0.1MOI, to cell culture after 1.5h
The HHT of various concentration is added in liquid, the activity of HHT is followed successively by 5,10,25,50,100nM from low to high.Virus infection is for 24 hours
Afterwards, it is observed in fluorescence microscope Olympus IX73, with CellSens software image, the results showed that, HHT significantly inhibits GFP-
NDV infects and is in dose dependent (Fig. 3 A).
HeLa cell is infected with the GFP-NDV of 0.1MOI, the HHT of various concentration is added after 1.5h into cell culture fluid,
Cell is collected after virus infection 36h, Western blot detects virus protein N P expression, the results showed that, HHT significantly inhibits NDV egg
It is white to express and be in dose dependent (Fig. 3 B).
HeLa cell is infected with the GFP-NDV of 0.1MOI, the HHT of various concentration is added after 1.5h into cell culture fluid,
Virus infection for 24 hours after, observed in fluorescence microscope Olympus IX73, statistics HHT inhibiting rate that GFP-NDV is infected, as a result
Show that HHT is 18nM (Fig. 3 C) to the half inhibiting rate of GFP-NDV.
11 age in days SPF chicken embryos are inoculated with the GFP-NDV of 50PFU, experimental group injects the HHT of 0.07,0.1,0.2mg/kg,
36h, 48h harvest chicken embryo, after 4 degree placements 6h, collect allantoic fluid, measurement hemagglutinative titer, statistics virus titer, the results show that phase
Compared with experimental group, 0.2mg/kg experimental group significantly inhibits NDV hemagglutinative titer (Fig. 3 D).
40 age in days SPF chickens are inoculated with the NDV of 10000PFU, the HHT of experimental group intramuscular injection 0.2mg/kg continuously injects 3
It, takes chicken whole blood, liver, lung for the 7th day after virus inoculation, and RT-PCR method detects the mRNA level in-site of virus protein N in whole blood, statistical number
According to the results show that HHT significantly inhibits NDV in the intracorporal proliferation (Fig. 3 E) of chicken.
Influence of 3 HHT of embodiment to PEDV virus virulence
The HHT of various concentration is added after the PEDV of Vero cell infection 0.1MOI, 1.5h into cell culture fluid, after 48h
Cell is harvested, Western blot analyzes viral protein expression, the results showed that, the inhibiting rate of 100nM HHT is 80% (Fig. 4 A).
The PEDV of Vero cell infection 0.1MOI, before PEDV infects 2h, infection simultaneously, after infection after 2h, infection
6h adds the HHT of 50nM, and after infecting 48h, indirect immunofluorescence method detects virus protein N protein fluorescence signal, the results showed that, sense
2h is added HHT and does not significantly inhibit virus replication before contaminating, and infects simultaneously, 6h adds HHT to significantly inhibit disease after 2h and infection after infection
Poison duplication (Fig. 4 B).
The PEDV of Vero cell infection 0.1MOI, infection while the HHT for adding various dose after infecting 48h, measure virus drop
Degree, the results showed that, 500nM HHT completely inhibits virus replication (Fig. 4 C).
3 age in days Infection in Piglets PEDV, experimental group with each three groups of control group, experimental group infection is administered simultaneously HHT, concentration is
0.05mg/kg, continuous injection adopt whole blood after 2 days, 5 days and carry out RT-PCR detection, measure the mRNA water of serum inner virus albumen NP
It is flat, the results show that HHT significantly inhibits PEDV is proliferated (Fig. 4 D) in the piglet.
Influence of 4 HHT of embodiment to HSV-1 virus virulence
With the HSV-1 vero cells infection of 0.1MOI, the HHT of various concentration, disease is added after 1.5h into cell culture fluid
Cell is collected after poison infection 48h, Western blot detects the expression and cytopathy of virus protein ICP8 albumen, as a result table
Bright, HHT significantly inhibits HSV-1 viral protein expression and virion generates, and is in dose dependent (Fig. 5).
Influence of 5 HHT of embodiment to AIV virus virulence
11 age in days SPF chicken embryos are inoculated with the AIV of 50PFU, experimental group injects the HHT of 0.1mg/kg, and 48h, 72h harvest chicken
Embryo after 4 degree of placement 6h, collects allantoic fluid, measures hemagglutinative titer, counts virus titer, the results showed that, HHT can reduce AIV blood clotting
10 times of potency (Fig. 6).
Influence of 6 HHT of embodiment to PRRSV virus virulence
With the PRRSV infection Marc145 cell of 0.1MOI, the HHT of various concentration is added after 1.5h into cell culture fluid,
Cell is collected after virus infection 36h, Western blot detects the expression of virus protein M albumen, the results showed that, HHT is significantly inhibited
PRRSV viral protein expression and be in dose dependent (Fig. 7).
Influence of 7 HHT of embodiment to TGEV virus virulence
PK15 cell is infected with the TGEV of 0.1MOI, the HHT of various concentration, virus is added after 1.5h into cell culture fluid
Cell is collected after infection 48h, Western blot detects the expression of virus protein N albumen, the results showed that, HHT significantly inhibits TGEV
Viral protein expression and be in dose dependent (Fig. 8).
Influence of 8 HHT of embodiment to FMDV virus virulence
PK-15 cell is infected with the FMDV of 0.1MOI, the HHT of various concentration, disease is added after 1.5h into cell culture fluid
Cell is collected after poison infection 6h, Western blot detects the expression of virus protein VP0, VP1, VP2, VP3 albumen, the results showed that,
HHT significantly inhibits FMDV viral protein expression and in dose dependent (Fig. 9).
Influence of 9 HHT of embodiment to PRV virus virulence
The HHT of various concentration, virus sense are added after 1.5h into cell culture fluid for the PRV vero cells infection of 0.1MOI
Cell is collected after dye 36h, Western blot detects the expression of virus protein gC albumen；After infection 48h freeze thawing three times, using cell
Lesion method measures virus virulence, the results showed that, HHT significantly inhibits PRV viral protein expression and virion generates, and is in dosage
Dependence (Figure 10).
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. being originated from application of the alkaloid component of cephalotaxus plant in preparation livestock and poultry broad-spectrum antiviral preparation.
2. application according to claim 1, which is characterized in that the alkaloid component includes HHT, cephalotaxine, tricuspid
China fir ester.
3. application according to claim 1 or 2, which is characterized in that the virus includes Paramyxoviridae, orthomyxovirus
Section, coronaviridae, Rhabdoviridae, herpetoviridae, Picornaviridae and Arteriviridae virus.
4. application according to claim 3, which is characterized in that it is described virus include VSV, NDV, PEDV, TGEV, AIV,
HSV-1、PRV、PRRSV、FMDV。
5. the plant source preparation of livestock and poultry viral infection resisting, which is characterized in that active constituent is to separate from cephalotaxus plant
Alkaloid component, including HHT, cephalotaxine, cepehalotaxus fortunei ester.
6. livestock and poultry anti-viral pharmaceutical compositions, which is characterized in that comprising the alkaloid component separated from cephalotaxus plant,
And at least one pharmaceutical excipient；The alkaloid component includes HHT, cephalotaxine, cepehalotaxus fortunei ester.
7. preparation according to claim 5 or pharmaceutical composition as claimed in claim 6, which is characterized in that the preparation
Or pharmaceutical composition is intramuscular injection, the subcutaneous or dosage forms such as oral.
8. preparation according to claim 5 or pharmaceutical composition as claimed in claim 6, which is characterized in that the administration of HHT
Amount is 0.01-1mg/kg weight.
9. preparation according to claim 5 or pharmaceutical composition as claimed in claim 6, which is characterized in that the virus
Including Paramyxoviridae, orthomyxoviridae family, coronaviridae, Rhabdoviridae, herpetoviridae, Picornaviridae and artery
The virus of scorching Viraceae.
10. preparation according to claim 5 or pharmaceutical composition as claimed in claim 6, the virus include VSV,
NDV、PEDV、TGEV、AIV、HSV-1、PRV、PRRSV、FMDV。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109745328A (en) * | 2019-03-11 | 2019-05-14 | 中国农业科学院兰州兽医研究所 | A kind of application of homoharringtonine in the drug of preparation prevention mouth disease virus infection |
| WO2021196666A1 (en) * | 2020-03-30 | 2021-10-07 | 中山大学 | Use of homoharringtonine in preparation of human body beta-coronavirus replication inhibitor |
| CN114177184A (en) * | 2021-12-06 | 2022-03-15 | 西安交通大学 | Application of harringtonine in preparing medicine for resisting novel coronavirus |
-
2017
- 2017-08-21 CN CN201710719849.8A patent/CN109419804B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109745328A (en) * | 2019-03-11 | 2019-05-14 | 中国农业科学院兰州兽医研究所 | A kind of application of homoharringtonine in the drug of preparation prevention mouth disease virus infection |
| WO2021196666A1 (en) * | 2020-03-30 | 2021-10-07 | 中山大学 | Use of homoharringtonine in preparation of human body beta-coronavirus replication inhibitor |
| US11850250B2 (en) | 2020-03-30 | 2023-12-26 | Hangzhou Minsheng Pharmaceutical Co., Ltd. | Use of homoharringtonine in preparation of betacoronavirus replication inhibitor in human |
| CN114177184A (en) * | 2021-12-06 | 2022-03-15 | 西安交通大学 | Application of harringtonine in preparing medicine for resisting novel coronavirus |
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